High spatial resolution imaging of a structure of interest in a specimen

ABSTRACT

For the high spatial resolution imaging of a structure of interest in a specimen, a substance is selected from a group of substances which have a fluorescent first state and a nonfluorescent second state; which can be converted fractionally from their first state into their second state by light which excites them into fluorescence, and which return from their second state into their first state; the specimen&#39;s structure of interest is imaged onto a sensor array, a spatial resolution limit of the imaging being greater (i.e. worse) than an average spacing between closest neighboring molecules of the substance in the specimen; the specimen is exposed to light in a region which has dimensions larger than the spatial resolution limit, fractions of the substance alternately being excited by the light to emit fluorescent light and converted into their second state, and at least 10% of the molecules of the substance that are respectively in the first state lying at a distance from their closest neighboring molecules in the first state which is greater than the spatial resolution limit; and the fluorescent light, which is spontaneously emitted by the substance from the region, is registered in a plurality of images recorded by the sensor array during continued exposure of the specimen to the light.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of International PatentApplication PCT/EP2007/003714 entitled “Method and Fluorescent LightMicroscope for the High-Resolution Three-Dimensional Representation ofthe Structure of a Specimen”, which was filed on 27 Apr. 2007 and claimsthe priority of German Patent Application No. DE 10 2006 021 317.3entitled “Verfahren und Fluoreszenzlichtmikroskop zum räumlichhochauflösenden Abbilden einer Struktur einer Probe” [Method andfluorescent light microscope for the high spatial resolution imaging ofa structure of a specimen], which was filed on 6 May 2006 and which ledto German Patent DE 10 2006 021 317.3 of 11 Oct. 2007.

FIELD OF THE INVENTION

The invention relates to a method for the high spatial resolutionimaging of a structure of interest in a specimen. More precisely, theinvention relates to a method for the high spatial resolution imaging ofa structure of interest in a specimen, having the steps: selecting asubstance from a group of substances which can be converted repeatedlyby a switching signal from a first state having first optical propertiesinto a second state having second optical properties, and which canreturn from the second state into the first state; marking thespecimen's structure of interest with molecules of the substance;applying an intensity of the switching signal to the specimen in orderto convert fractions of the substance into the second state by theswitching signal; imaging the specimen onto a sensor array; using thesensor array to register an optical signal which comes from the fractionof the substance respectively remaining in the first state, in order torecord a distribution of the measurement signal over the sensor array.

BACKGROUND OF THE INVENTION

A method for the high spatial resolution imaging of a structure ofinterest in a specimen with the steps specified above, which is referredto as RESOLFT (REversible Saturable OpticaL Fluorescence Transition), isknown from US 2004/0212799 A1 and US 2006/0038993 A1. Here, whenconverting the substance into the second state by the switching signal,only a defined spatial region of the specimen is respectively omitteddeliberately. This region is an intensity minimum of an interferencepattern with a zero position, and the intensity of the switching signalis already so large everywhere in the vicinity of the zero position thatit exceeds a saturation threshold for complete switching of thesubstance into the second state. In this way an optical measurementsignal, which comes from the fraction of the substance remaining in thefirst state, can be assigned to the specimen's region deliberatelyomitted by the switching signal. The spatial resolution for imaging thespecimen's structure of interest, which is marked with the substance,therefore no longer depends on the spatial resolution limit of theimaging of the specimen onto the sensor array being used. Rather thespatial resolution is defined by the extent of the zero position of theswitching signal, within which the substance still lies in the firststate, since there is no measurement signal which can come from thevicinity of the zero position and accordingly needs to be assigned to aspatial position separable from the position of zero position. Whenspatially imaging the structure of interest in a specimen, it istherefore possible to go below the resolution limit (i.e. the Abbe limitdue to diffraction, which is given by the wavelength of the lightdivided by two times the numerical aperture) which in principlerestricts the spatial resolution of the imaging optical methods anddepends directly on the wavelength of the longest-wave relevant opticalsignal.

The substances used in the above-described RESOLFT method for markingthe structure of interest in the specimen are switchable fluorescentdyes. This is explained in US 2004/0212799 A1 and US 2006/0038993 A1 inthat they are selected from a group of substances which can be convertedrepeatedly by a switching signal from a first state having first opticalproperties into a second state having second optical properties, andwhich can return from the second state into the first state, the twostates differing at least in respect of one of the following criteria:conformational state of a molecule; structural formula of a molecule;spatial arrangement of atoms within a molecule; spatial arrangement ofbonds within a molecule; accumulation of further atoms or molecules on amolecule; grouping of atoms and/or molecules; spatial orientation of amolecule; mutual orientation of neighboring molecules and orderingformed by a multiplicity of molecules and/or atoms.

The placement of the zero position of the switching signal within thespecimen can be determined from the intensity distribution of themeasurement signal over the sensor array with an accuracy higher thanthe spatial resolution limit of the imaging, if it is certain that themeasurement signal comes only from the region of this one zero position.Besides the size of the zero position, the accuracy achievable in thelocation determination essentially depends only on the density of thepixels of the sensor array, which is conventionally a CCD or CMOScamera, as well as the signal-to-noise ratio achieved and the width ofthe point spread function of the imaging. Specifically, the accuracyachievable for the location determination is even much finer than thespacing of the pixels of the sensor array divided by the imaging scale;with a good signal-to-noise ratio even much less than one nanometer,which is known to the person skilled in the art.

It is also known to use this phenomenon for the localization ofindividual fluorescent molecules in a specimen. A prerequisite for this,however, is that the individual fluorescent molecules lie at a distancefrom their respective closest neighboring molecules which is greaterthan the spatial resolution limit of the imaging of the specimen ontothe sensor array, since otherwise the optical measurement signalsreceived by the sensor array from the individual fluorescent moleculesmerge together. When this happens, the positions of the individualmolecules can no longer readily be determined.

In the method known from US 2004/0212799 A1 and US 2006/0038993 A1 andall previous methods in which a structure of interest in a specimen ismarked with a substance emitting a measurement signal, i.e. inparticular a fluorescent substance, the density of the molecules of thesubstance in the specimen is regularly so large that the distance of theindividual molecules from their closest neighbors corresponds only to asmall fraction of the spatial resolution limit of the specimen's imagingonto the sensor array. WO 2006/127692 A2 has disclosed a method for thehigh spatial resolution imaging of a structure of interest in aspecimen, in which the structure of interest is marked with switchablefluorescent dyes in the form of so-called phototransportable opticalmarkings. A subgroup of the markings is respectively activated into astate in which they can be excited to emit fluorescent light. Therespective subgroup comprises so few of the markings that they lie at adistance from one another which is greater than the spatial resolutionlimit for imaging the specimen onto the sensor array. This makes itpossible, after exciting the markings of the subgroup into fluorescence,to localize the origin positions of the fluorescent light with aresolution better than the diffraction limit which applies for thespatial resolution for imaging the specimen onto the sensor array, sothat a point of the marked structure of interest is also respectivelyrecorded with this increased resolution. The phototransformable opticalmarkings are defined in WO 2006/127692 in that they can be switched onby an activating signal into a state in which they can be excited toemit fluorescent light. This activating signal may be the same as theexcitation light which subsequently excites the markings intofluorescence. More specific embodiments of phototransformable opticalmarkings, which are disclosed in WO 2006/127692, comprise exclusively aphotoactivatable fluorescent proteins, i.e. molecules which become afluorophore only after they have absorbed at least one light quantum, orin other words they initially need to be switched on before they arefluorescent. The-activating or switching process entails a modificationof the molecular structure of the molecules (relocation of atom groupsor even breaking or forming a bond). The method known from WO2006/127692 is also referred to as PALM (Photoactivated LocalizationMicroscopy).

A similar method known as STORM (Stochastic Optical ReconstructionMicroscopy) and described by Rust et al. in Nature Methods, 3, 793-796(2006) likewise uses molecules switchable into a fluorescent state, i.e.switchable fluorescent dyes, although these are not proteins butphotoswitchable organic fluorophores, specifically the fluorescent dyesCy3 and Cy5. It is known of these cyanine dyes that they can be switchedbetween different conformational states, more specifically isomericstates.

A disadvantage of the PLM and Storm methods is that it is not possiblein them to predict when the structure of interest in the specimen willbe recorded so fully that determining the position of further moleculesprovides no additional useful information and the method may thereforebe terminated.

The range of switchable proteins and fluorophores, which may be used forthe RESOLFT, PALM and STORM methods explained above, is very smallcompared with the total number of fundamentally known and availablefluorescent dyes. Dyes which are both switchable and (in one of theswitching states) capable of fluorescence, are very rare. They aretherefore synthesized and optimized by elaborate methods. Added to this,the switching behavior and the fluorescent behavior depend very stronglyon the chemical environment of the molecule. This applies both forswitchable fluorescent proteins and for switchable organic fluorophores.This deficiency is to be regarded as fundamental, and it is associatedinter alia with the fact that fluorescence and switching of the moleculeare mutually competitive molecular processes which often compete withone another from the same excited state. The brightness of theswitchable fluorescent dyes in their fluorescent state, i.e. therelative yield of fluorescent light from a molecule during repeatedexcitation, is also often only small compared with a multiplicity ofnonswitchable organic fluorophores and nonswitchable fluorescentproteins. The strong restrictions due to switchable proteins orfluorophores, however, have to date being tolerated in order to obtainthe high spatial resolutions achievable by the aforementioned methodsfor imaging structures of interest.

In so-called GSD (Ground State Depletion) microscopy (S. Bretschneideret al.: Breaking the diffraction barrier in fluorescence microscopy byoptical shelving, Phys. Rev. Lett. 98, 218103 (2007)), the diffractionlimit for imaging a structure marked by a fluorescent dye in a specimenis overcome by converting the respective fluorescent dye outside therespective measurement point from its electronic ground. state, fromwhich it can be excited into fluorescence by excitation light, into adark electronic state in which it is not capable of fluorescence. Thisis done before exciting the remaining molecules at the measurement pointinto fluorescence by depopulating light with the same wavelength as theexcitation light. The dark electronic state is typically a tripletstate, while the ground state of the fluorescent dye is a singlet state.The molecules typically return thermally, i.e. not (optically) switched,from this dark state into the electronic ground state, so that onlylight of a single wavelength i.e. the excitation light is necessary forcarrying out the experiment.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a method for the highspatial resolution imaging of a structure of interest in a specimen,having the steps: selecting a substance from a group of substances whichcan be converted repeatedly by a switching signal from a first statehaving first optical properties into a second state having secondoptical properties, and which can return from the second state into thefirst state; marking the specimen's structure of interest with moleculesof the substance; applying an intensity of the switching signal to thespecimen in order to convert fractions of the substance into the secondstate by the switching signal, the intensity of the switching signalbeing set so that at least 10% of the molecules of the substancerespectively remaining in the first state lie at a distance from theirclosest neighboring molecules in the first state which is greater thanthe spatial resolution limit of the imaging of the specimen onto thesensor array; imaging the specimen onto a sensor array, a spatialresolution limit of the imaging being greater (i.e. worse) than anaverage spacing between closest neighboring molecules of the substancein the specimen; using the sensor array to register an optical signalwhich comes from the fraction of the substance respectively remaining inthe first state, in order to record a distribution of the measurementsignal over the sensor array; and determining the position in thespecimen of the molecules of the substance respectively remaining in thefirst state, which lie at a distance from their closest neighboringmolecules in the first state which is greater than the spatialresolution limit of the imaging of the specimen onto the sensor array,from the distribution of the measurement signal over the sensor array.

A second aspect of the present invention provides a method for the highspatial resolution imaging of a structure of interest in a specimen,having the steps: selecting a substance from a group of substances,which have a first state with first fluorescent properties and a secondstate with second fluorescent properties; which can be excited by lightof one wavelength to spontaneously emit fluorescent light; which can beconverted from the first state into their second state by the light ofthe one wavelength and which can return from their second state intotheir first state; marking the specimen's structure of interest withmolecules of the substance; imaging the specimen onto a sensor array, aspatial resolution limit of the imaging being greater (i.e. worse) thanan average spacing between closest neighboring molecules of thesubstance in the specimen; exposing the specimen to the light of the onewavelength in a region which has dimensions larger than the spatialresolution limit of the imaging of the specimen onto the sensor array,fractions of the molecules of the substance alternately being excited bythe light of the one wavelength to spontaneously emit fluorescent lightand being converted into their second state, and at least 10% of themolecules of the substance that are respectively in the first statelying at a distance from their closest neighboring molecules in thefirst state which is greater than the spatial resolution limit of theimaging of the specimen onto the sensor array; registering thefluorescent light which is spontaneously emitted from the region ofmolecules of the substance, in a plurality of images recorded by thesensor array during continued exposure of the specimen to the light ofthe one wavelength; and determining the position in the specimen of themolecules of the substance that are respectively in the first state,which lie at a distance from their closest neighboring molecules in thefirst state which is greater than the spatial resolution limit of theimaging of the specimen onto the sensor array, from the images recordedby the sensor array.

The two aspects of the present invention have substantial identicaloverlaps, even though they will sometimes be explained in more detailseparately below. In each case, the invention is based on a substancefor marking the structure of interest in a specimen, which delivers ameasurement signal in its first state in which it is normally found butnot in its second state, into which it can be converted.

When converting the fraction of the substance into the second state inthe method of the first aspect of the present invention, an intensity ofthe switching signal is set so that a substantial percentage of themolecules respectively remaining in the first state lie at a distancefrom their closest neighboring molecules in the first state which isgreater than the spatial resolution limit of the imaging of the specimenonto the sensor array. For each molecule of the substance which belongsto this substantial percentage, its position in the specimen can bedetermined from the intensity distribution over the sensor array of theoptical measurement signal coming from it, with the alreadyfundamentally known extremely high position resolution whichconsiderably surpasses the spatial resolution limit of the imaging ofthe specimen onto the sensor array. This moreover also allows thespecimen's structure of interest, marked with the substance, to beimaged with this extremely high position resolution. It is then onlynecessary for other molecules of the substance to be reset into thefirst state, these molecules belonging to the substantial percentage ofthe molecules of the substance in the first state in which they lie at adistance from their closest neighboring molecules in the first statewhich is greater than the spatial resolution limit of the imaging of thespecimen onto the sensor array. This represents no problem, however,that since the selection of the molecules for which this conditionapplies is based on transition probabilities and therefore obeysstatistical laws. Since statistically speaking, other molecules of thesubstance will tend to be switched into the second state, the specimen'sstructure marked with the substance will be interrogated withever-higher density according to the position of the individualmolecules of the substance. The sum of these positions is the image ofthe structure of interest, obtained by the new method, which has theposition resolution far finer than the conventional resolution limit.Admittedly, individual molecules may repeatedly belong to the selectionwhich satisfies the criterion applied. here. Yet precisely when only acomparatively small fraction of the molecules present overall in thespecimen's region in question is converted into the second state andfulfils the distance criterion, the fraction of molecules whose exactlocation may be determined repeatedly is negligible compared with thefraction of molecules which belong only once to the substantialpercentage for which accurate determination of the location is possible,even with frequent repetition of the conversion of the fraction of thesubstance into the second state. Even if this fraction is larger, atworst the efficiency is thereby impaired, i.e. in particular the speedbut not the function of the new method. This is due to the fact that byselecting the molecules for the position determination with maximalposition resolution based on transition probabilities, given asufficiently high number i.e. a sufficiently large random sample range,owing to statistical laws the ensemble of molecules whose positions aredetermined is representative of the ensemble of molecules of thesubstance which are present in the specimen and therefore the specimen'sstructure of interest marked with them.

In order to be able to determine the placement of the molecule in thespecimen from the intensity distribution over the sensor array of themeasurement signal coming from a molecule of the substance in the firststate, with a position resolution higher than the spatial resolutionlimit of the imaging of the specimen onto the sensor array, it is to beunderstood that the grid dimension of the pixels of the sensor arraymust at least be smaller than the spatial resolution limit times theimaging factor of the imaging. The greater dimensioning is preferablyonly at most half as great as the spatial resolution limit times theimaging factor of the imaging, so that the measurement signal comingfrom a molecule is distributed over at least four pixels of the sensorarray. A much smaller grid dimension of the pixels of the sensor arrayis not associated with a corresponding increase in the positionresolution when determining the placement of the molecules in thespecimen. Rather, it entails the risk that the signal-to-noise ratiowill become significantly worse. A grid dimension which lies between 60and 10% of the spatial resolution limit times the imaging factor of theimaging will therefore generally be most favorable.

The substantial percentage of the molecules respectively remaining inthe first state at a given time, which satisfies the criterion of thespacing of the molecules in the second state being larger than thespatial resolution of the imaging of the specimen onto the sensor array,is at least 10%. In particular when the percentage complementary theretoof the molecules in the first state which lie at a smaller distance fromone another is comparatively large, it is important that the measurementsignal coming therefrom or its intensity distribution over the sensorarray to be separated from the measurement signal which comes from themolecules at a sufficient distance from one another. This may be done bychecking whether the total intensity of the measurement signal, theshape and/or the area of the intensity distribution of the measurementsignal over the sensor array corresponds to a single molecule or aplurality of molecules. Only in the event of correspondence with asingle molecule is the location of this molecule then determined fromthe intensity distribution. It is to be understood that it is expedientto be able to determine locations of individual molecules of thesubstance, and therefore positions of the specimen's structure ofinterest, for a maximally large percentage fraction of the registeredlocal intensity distributions. For this reason, it is preferable for amaximally large percentage of the molecules respectively converted intothe second state to lie at the requisite large distance from neighboringmolecules in the second state. It makes little sense, however, toattempt to prevent all the molecules actually remaining in the firststate from having any neighbors which are likewise in the first stateand whose spacing is less than the spatial resolution limit of theimaging of the specimen onto the sensor array, because the averagespacing of the molecules which can respectively be in the second statewould thereby become very large, the effect of which would be that thenumber of locations of molecules which can be determined after aswitching process decreases again. The switching signal intensity withwhich the specimen's structure of interest can in the end be imaged mostrapidly, i.e. with the fewest repetitions of the switching process,finally depends on how difficult it is to distinguish between intensitydistributions of the measurement signal over the sensor array which areassignable to only one molecule in the first state or to two or moreneighboring molecules in the first state.

Since the new method makes do without spatial structuring of theswitching signal in the region of the specimen, the switching signal mayin principle also be a non-optical signal, although an optical switchingsignal is actually preferable owing to its simpler handleability. Withan optical switching signal, for example, different intensities of theswitching signal may readily be applied to different regions of thespecimen in order to accommodate the fact that the substance, with whichthe specimen's structure of interest is marked, is present in verydifferent concentrations in these various regions. Nevertheless, it willalways be the case that the intensity of the switching signal in the newmethod has a constant value over regions that have dimensions largerthan the spatial resolution limit of the imaging of the specimen ontothe sensor array. Specifically, this constant value may be set inverselyproportionally to an average local concentration of the substance inthis region of the specimen.

In order to establish rapidly whether the measurement signal coming froma region of the specimen, which corresponds to a plurality of pixels ofthe sensor array, is assignable to one or more molecules of thesubstance, so that it is viable to record an intensity distribution ofthe measurement signal in order to carry out a position determination ofan individual molecule therefrom, or not, this region may be imaged inparallel with the sensor array onto a photodetector by which thechronological sequence of the emission of individual photons from theregion can be observed. If only a single molecule in the region is inthe first state, the photons of the measurement signal which are emittedby it will have a minimum time spacing because the molecule canrespectively emit only one photon in a cycle of its excitation.Correspondingly, photons following one another more closely indicatethat they have been emitted by a plurality of molecules in the regionwhich are in the second state. In this case, the process of recordingthe intensity distribution of the measurement signal may be terminatedand a new attempt may be made to obtain only a single molecule of thesubstance in the first state in the region. The photodetector forobserving the chronological sequence of the emission of individualphotons from the region may comprise a single detector unit, if the deadtime after registering a first photon is sufficiently short. Otherwise,the photodetector should be constructed from two detector units withwhich a deliberate search is made for photons emittedquasi-simultaneously from the region in the scope of a coincidencedetector. These are an indication of a plurality of molecules of thesubstance in the region simultaneously being in the second state.

As already indicated in the preceding description of the invention, itrelates in particular to those embodiments of the new method with whichthe substance is selected from a subgroup of substances which in a firststate can be excited by an optical excitation signal to spontaneouslyemit fluorescent light, which is registered as an optical measurementsignal by the sensor array.

The substance may furthermore be selected from a subgroup of substanceswhich can be converted from the second state back into the first stateby an optical switchback signal. In this case, before converting anotherfraction of the substance into the second state by the switching signal,the fraction of the substance previously converted into the second statemay deliberately be converted back into the first state by theswitchback signal. It is not therefore necessary to wait for the timetaken by the molecules of the substance to return from the second stateinto the first state, for example owing to thermal excitation or underthe effect of the optical excitation signal, which is primarily intendedhere for the spontaneous emission of fluorescent light. If the half-lifeof the molecules of the substance in the second state has a favorableorder of magnitude, however, then the new method may also be carried outvery effectively when the substance is not convertible from the secondstate back into the first state by an optical switchback signal.

A particularly preferred group of substances for use in the new methodare so-called Förster resonance energy transfer pairs, conventionallyreferred to as FRET pairs for short, which consist of an (energy) donorand an (energy) acceptor. Primarily those FRET pairs in which theacceptor is photochromic and is switched by the switching signal, inorder to modulate fluorescence properties of the donor, are suitable foruse in the new method. The acceptor itself may be fluorescent, althoughthis will generally not be utilized in the new method.

In cases in which the structure of interest in the specimen is definedby a protein, it is advantageous for the substance, with which thestructure of interest is marked, to be incorporated into the molecule orattached to it by gene technology. The incorporation or attachment maybe carried out directly, i.e. the marking substance per se may beinserted into the protein in the specimen or appended to it in the scopeof a fusion protein. It is however also possible merely for a bindingsite for the marking substance, via which the marking with the substancecan then take place subsequently, to be inserted into the protein orappended to it. Such a widely known binding site for fluorescentproteins for marking a protein consists, for example, of the sequence of4 cysteines in the amino acid sequence of the protein, i.e. a so-calledtetracysteine motif. It is particularly advantageous for thegene-technological incorporation or attachment of the substance, or thebinding site for the substance, into or onto the protein in the specimento be carried out by expression of the protein modified in this wayinstead of the original protein in at least one cell within thespecimen, so that the structure of interest is formed directly by theprotein modified with the marking substance or with the binding sitesfor it.

The extremely high position resolution for location determination offersthe opportunity to observe the spatial arrangement of very smallstructures in the specimen. Such a structure may consist of a singleprotein molecule, which is then to be marked with the substance at aplurality of different points. These points very typically lie at adistance from one another which is less than the spatial resolutionlimit of any conceivable optical imaging of the protein. In the newmethod, the measurement signal of the substance is respectivelyinterrogated at a given time at only one of the points where the proteinmolecule is marked with the substance. With each repetition of theinterrogation, the substance is switched over by the switching signal atonly one of the marking points into the second state which delivers themeasurement signal. This may admittedly be the same point as before. Yetsince this process is controlled by transition probabilities, with asufficiently large number of repetitions of the interrogation it mayreliably be assumed that the substance has been interrogated at allmarking points of the protein molecule and that the position of allmarked points of the protein molecule has therefore been determined witha maximal position resolution. The position resolution is in this caseso large that even protein foldings and other conformational changes ofprotein molecules become observable.

The total time required for imaging the structure of interest in aspecimen can be shortened by carrying out the new method in parallelwith at least two different substances, from which distinguishableoptical measurement signals come in their respective first state. Thespeed of the new method is limited by the fact that the density of themolecules of the substance in the first state which lie at a sufficientdistance from one another, and therefore the number of molecules whoselocations can be determined in a cycle of the new method, is naturallyrestricted. So long as the optical measurement signals of two differentsubstances marking the structure of interest differ so that they can bemutually separated, by using such different substances the number ofmolecules whose locations can be determined in a cycle of the new methodcan be increased proportionally to the number of different substances.

A specific embodiment of the method of the first aspect of the presentinvention, known as PALMIRA (PALM with Independently RunningAcquisition), is described in C. Geisler, A. Schönle, C. vonMiddendorff, H. Bock, C. Eggeling, A. Egner and S. W. Hell: Resolutionof λ/10 in fluorescence microscopy using fast single moleculephoto-switching, Appl. Phys. A 88, 223-226 (2007), the disclosure ofthis publication being fully incorporated here by reference. In thisembodiment of the method according to the invention, a structure ofinterest in a specimen is marked with a switchable fluorescent protein.Specifically this is a protein by the name of rsFastLime, which by alight with a wavelength of 488 nm is not only excited into fluorescencein its initial state but also fractionally switched off into anonfluorescent state and partially switched back again therefrom intoits fluorescent state. The underlying mechanism is a conformationalchange of the fluorophore. These properties of the switchablefluorescent protein make it possible, with the light of only a singlewavelength, alternately to set up subgroups of fluoresceable moleculesof the protein in which the fluoresceable molecules lie at a mutualspacing greater than the diffraction limit, and to excite thefluoresceable molecules into fluorescence. It is thereby possiblecontinuously, i.e. with a high frequency, for images to be recordedwhich register the alternating subgroups of the fluorescent moleculesand in which the position of the respectively registered molecule can bedetermined with an accuracy beyond the diffraction limit. With the sumof the images, the structure in the specimen is recorded with a spatialresolution finer than the diffraction limit.

At the start of the exposure of the specimen to the light of the onewavelength in the method of the second aspect of the present invention,its intensity is set so that the substance is converted from itsfluorescent first state into its nonfluorescent or at leastdistinguishably fluorescent second state until at least 10%, preferablyat least 50%, more preferably at least 90% and most preferably at least95% of the molecules of the substance which remain in the first statelie at the critical distance from their closest neighboring molecules inthe first state, which is greater than the spatial resolution limit ofthe imaging of the specimen onto the sensor array. This is generallyequivalent to saying that more than 90%, preferably more than 95%, evenmore preferably more than 98% and most preferably more than 99% of themolecules of the substance are converted into the second electronicstate. Images, in which the fluorescent light spontaneously emitted bythe substance is registered, are subsequently recorded by the sensorarray while the specimen continues to be exposed to the light of the onewavelength. The effect of the intensity of the light being reduced ifneed be relative to the initial intensity, is that the molecules whichare respectively still in the first state are excited into spontaneousemission of fluorescent light which is registered in the images, and toa smaller part are converted into the nonfluorescent or differentlyfluorescent first state. This smaller portion of the molecules of thesubstance is ideally precisely as large as a fraction of the moleculesof the substance which simultaneously return from the second state intothe first state spontaneously or under the effect of the same light ofthe one wavelength. The images recorded continuously with the exposureof the specimen to the light of the one wavelength thus always registerfluorescent light from as many isolated molecules of the substance ascan have their position determined in the specimen with an accuracyfiner than the diffraction limit. From the sum of the individualpositions registered in this way, the structure in the specimen which ismarked with the substance can likewise be recorded with a spatialresolution finer than the diffraction limit.

It is not always necessary to reduce the intensity of the light of theone wavelength for recording the images; rather, this is necessary onlywhen the initial concentration of the fluorophores was very great. Formedium and low concentrations, the intensity can be adjusted from thestart so that the isolation and registering of the molecules can becarried out with the same value of the intensity. This is related to thefact that the number of photons which are emitted successively asfluorescent light by the fluorescent dye in the first state untilconversion into the second state (in the same fluorescence burst) issubstantially independent of the intensity of the light being used.

It is extremely surprising that even though it has essentially the sameprocedure as the method described as PALMIRA, the method of the secondaspect of-the invention makes do without switchable proteins orfluorophores. Instead, as a fluorescent dye in the new method, asubstance may be used in which the first and second states are differentelectronic states of the substance, i.e. states of the substance whichdiffer from one another only in electronic terms. The substance thendoes not fall within the definitions of the substance used for themarking in US 2004/0212799 A1 and US 2006/0038993 A1, and it may be anyconventional non-switchable fluorescent dye. Besides their electronicground state from which they can be excited into fluorescence,practically all conventional fluorescent dyes have an electronic darkstate into which they can be converted at a relevant rate owing toexcitation with light of the same wavelength as can be used to excitethe fluorescence. These are generally a singlet state as thefluoresceable ground state and a triplet state as the dark state. Innormal fluorescent light microscopy, the fractional conversion of afluorescent dye into its nonfluorescent triplet state instead of itsexcited singlet state—especially with high intensities of the excitationlight—is known as a disadvantage because it reduces the yield offluorescent light from a specimen. In the present invention, this effectis specifically utilized because the position of any individual moleculeof the substance, with which the structure of interest in the specimenis marked, be recorded with a resolution better than the diffractionlimit only when the fluorescent light from the molecule can beregistered in isolation, i.e. separately from the fluorescent light ofneighboring molecules. To this end, only few of the molecules shouldrespectively be in the first state.

In the new method of the second aspect of the present invention, it isfrequently advantageous to implement measures which modify the lifetimeof the nonfluorescent second state of the fluorescent dye in thespecimen. In contrast to conventional fluorescence microscopy, however,this often involves not extending but shortening the lifetime of thenonfluorescent second state. The measures which cause such shortening ofthe lifetime include cooling the specimen to low temperatures at whichthermal excitations are reduced to collision-induced transitions,reducing the concentration in the specimen of oxygen which quenches thetriplet state of the fluorescent dye, for example with a glucose oxidasewhich binds oxygen or by measurements in a vacuum, or fixing orembedding the specimen in polymers, for example PVA. The increasedlifetime of the nonfluorescent second state makes it possible to keeplarge fractions of the fluorescent dye in the nonfluorescent secondstate even with lower intensities of the light of the first wavelength.

The known risk of photobleaching a conventional fluorescent dye from itstriplet state likewise represents no problem in the new method. Strictlyspeaking, it is sufficient for a substantial fraction of the moleculesof the fluorescent dye to return once into their fluorescent first stateafter they have been pumped into their nonfluorescent second state ordifferently fluorescent second state. After this return, the moleculesare registered individually. Their subsequent fate is insignificant. Forinstance, they may enter the triplet state again and be photobleachedtherefrom.

Partial photobleaching of the fluorescent dye may even be carried outdeliberately in the new method of the second aspect of the presentinvention, before the remaining unbleached molecules are registered. Thenew method can be carried out particularly advantageously when themolecules of the fluorescent dye do not exceed a particular spatialdensity in the specimen, because then a particular percentage of themolecules which remains in the first state likewise does not exceed aparticular spatial density, which is essential for being able toregister the individual molecules separately. If the actualconcentration of the fluorescent dye in the specimen exceeds theparticular spatial density, on the other hand, it may be difficult toregister the molecules individually. In order to avoid this difficulty,the excess fluorescent dye may be switched off by photobleaching bymeans of a high intensity of the light of the one wavelength or anotherwavelength, i.e. converted into a persistent dark state which differsfrom the first state and the second state. The persistent dark state, inwhich the fluorescent dye is no longer involved in the steps ofrecording the images for registering the individual molecules, typicallydiffers not only electronically but for example also chemically from thefirst state and the second state which are used according to theinvention for this registering of the individual molecules.

The new method of the second aspect of the present invention was carriedout successfully with commercially available fluorescent dyes known asnon-switchable to any person skilled in the art, such as Rhodamine 6G.Compared with conventional fluorescent light microscopy using thisfluorescent dye—apart from different preferred details in the specimenpreparation—in order to be carried out the intensity of the light of theone wavelength merely needs to be tuned with the frequency at which theimages. are recorded by the sensor array. The equipment requirementsnecessary for this are available in many fluorescent light microscopes.Here, it is only necessary to modify the control of the intensity of thelight of the one wavelength according to the method according to theinvention. As an alternative, the frequency of the image recording bythe sensor array or the camera comprising the sensor array is altered.Accordingly, the new fluorescent light microscope is distinguished onlyby a special design of the control for the intensity of the light of theone wavelength. Preferably, online image processing is in this caseprovided for the individual images recorded by the sensor array.

This evaluation is expedient in order to adjust the intensity of thelight of the one wavelength to such a value which actually makes itpossible to register fluorescent light of individual molecules,spatially separated from one another, in the individual images. Thevalues set for the intensity of the light of the one wavelength may be aconstant value. This also includes a very fast pulse sequence with afrequency very much higher than the image frequency of the recordedimages. The intensity of the light of the one wavelength may howeveralso have an intensity profile temporally modulated with the sequence ofthe recording of the images, for example in order to deliberately set upthe subgroup of the molecules of the substance which are in the firststate, between the individual images, and to excite primarily themolecules of the set up subgroup into fluorescence during the recordingof the individual images. Furthermore, the light of the one wavelengthmay in this case be directed on to the respective region of interest inthe continuously (with the time-modulated intensity profile) or inpulses which are not resolved in the recording of the images (likewisewith the time-modulated intensity profile).

The online evaluation of the individual recorded images may be used todetermine the spatially inseparable fluorescent molecules of thesubstance, whereupon the intensity of the light may be varied until adensity threshold for such inseparable fluorescent molecules is fallenbelow. In this way, an upper limit for the intensity of the light of theone wavelength is defined. A lower limit may be defined in that theindividual recorded images can be evaluated online with respect to themaximal density with which they show separable fluorescent molecules ofthe substance, and in that the intensity of the light of the onewavelength is varied until a density threshold for such separablefluorescent molecules is reached from below. While on the one hand it isimportant for the molecules in the fluorescent state not to have aconcentration so high that they can no longer be registered separatelyfrom one another, their concentration below this limit should be as highas possible so as to obtain as much information as possible about thestructure of interest with each image.

The initial exposure of the substance to the light of the onewavelength, which is primarily used to convert them essentially intotheir second state, may also be used to record an intensity distributionof the fluorescent light of all the substance in the specimen by thesensor array. This intensity corresponds to a concentration distributionof the substance in the specimen with the spatial resolution of theimaging of the specimen onto the sensor array.

This concentration distribution of the substance in the specimenrepresents an overview of the position of the structure of interestmarked with the substance in the specimen. This simplifies the furthersteps of the new method, since it can thus for example be concentratedon to those regions of the specimen in which parts of the markedstructure are actually present. This is usually not possible withswitchable and above all activatable fluorophores since they areinitially for the most part not in the fluorescent state, whichprohibits an overview owing to the lack of signal.

Depending on the concentration distribution of the substance in thespecimen, the intensity of the light of the one wavelength may also beadjusted for the region respectively to be examined in more detail, orat least it may be preset to an approximately suitable value for thefine adjustment. Furthermore, a local termination criterion for therecording of further images of the same region of the specimen may bedefined on the basis of the concentration distribution of the substancein the specimen. The information content of additional images of aregion of the specimen comprises a decreasing information content, thedecrease in the information content depending on the concentration ofthe substance in the respective region. If only very few molecules ofthe substance are present in a region, then relatively few images aresufficient in order to record the position of a high percentage of themolecules. Further images contribute only redundant information in thisregard. The situation is different with a very high concentration of thesubstance in a region. Here, only smaller fractions of the substance inthe specimen are recorded even with many images, and each further imagemakes new information available.

Specifically, in the new method, each position of a molecule asregistered in the successive images is entered not only into a highresolution overall image of the structure of interest in the specimenbut, convoluted with the PSF (Point Spread Function) of the imaging ofthe specimen onto the sensor array, also into a reconstruction of theinitially recorded intensity distribution. When this reconstruction hasapproximated the initially recorded intensity distribution to within aparticular degree, no significant further information about thestructure of interest is to be expected with the positions of furthermolecules from further images. For the convolution with the PSF, thebrightness of the respective molecule may be taken into account as aweighting factor. Various values may be adopted as a measure of thesimilarity of the reconstruction to the initially recorded intensitydistribution, for example a cross correlation, a simple difference orquadratic deviation of the normalized intensity distributions ordeviations between the spatial frequencies (Fourier transforms) of theintensity distributions.

The new method is particularly well suited for marking the specimen'sstructure of interest with the non-switchable fluorophores by modifyinga biological specimen with gene technology so that it itself expressesthe non-switchable fluorescent dyes or specific binding sites for thenon-switchable fluorescent dyes or for linkers coupled thereto. Thestructure of interest in the specimen is particularly advantageouslymarked in this way with non-switchable organic dyes via so-called smalllabels or self-labeling protein tags such as FlAsh, snap tags or halotags. These and similar concepts are fundamentally known to the personskilled in the art, see for examplehttp://www.promega.com/cnotes/cn011/cn011_(—)02.htm orhttp://www.covalys.com/ or BioForum Europe 6, 51-59 (2005). This makesit possible to image proteins in a biological specimen with highresolution using widespread conventional fluorescent dyes.

For the fluorescent dye which is employed in the new method, it is notcrucial for its second electronic state to be nonfluorescent, i.e. notcapable of fluorescent and therefore entirely dark. It may also bedifferently fluorescent than the first electronic state. If thefluorescent dye is in this case excited into fluorescence in the secondstate by the same light as in its first electronic state, it isimportant that the fluorescent light which is emitted by the fluorescentdye in its first electronic state can be distinguished from thefluorescent light which is emitted by the fluorescent dye in the secondelectronic state.

It is to be understood that the new method may be combined with variousmeasures which are familiar to the person skilled in the art, inparticular from the field of methods known as PALM and STORM. Thesecomprise in particular measures for three-dimensional resolution of theregistered positions of the molecules in the specimen, i.e. for spatialresolution of these positions in the z direction as well. These measuresinclude multi-photon excitation of the fluorescent dye from its firststate, both for fluorescence and for transition into its nonfluorescentsecond state by focusing the exciting light of the one wavelength ontothe respective plane of interest, and using two mutually opposingobjectives with high numerical aperture in 4-pi configuration forexposing the specimen to the light of the one wavelength and/or forregistering the fluorescent light from the specimen. In so far as thelight is then respectively focused only into one or more individualpoints of the plane, the plane with these points is to be scanned in allsteps of the method, for example during the recording of each individualimage. The focusing of the light of the one-wavelength into individualpoints of the specimen may advantageously be combined with confocalregistering of the fluorescent light from the specimen. As analternative the specimen may be exposed, orthogonally to the directionof the imaging of the specimen onto the sensor array, to the light ofthe one wavelength from which a light section is formed by a cylindricallens. This procedure is known to the person skilled in the art as SPIM(Selective Plane IlluMination).

A fluorescent light microscope for carrying out the new method differsfrom known fluorescence microscopes, with which a spatial resolutionbetter than the diffraction limit is achieved, by the fact that thereare no measures for finely structuring the switching signal from theswitching signal source; rather, a control of the switching signalsource adjusts an intensity of the switching signal according to the newmethod instead.

At least one photodetector may additionally be provided, onto which aregion of the specimen that corresponds to a plurality of pixels of thesensor array is imaged, in order to observe the chronological sequenceof the emission of individual photons from this region. As alreadyexplained in connection with the new method, with such a photodetectorit is possible to establish very rapidly, i.e. in particular even beforethe readout of the sensor array, whether the intensity of themeasurement signal of only one or more molecules of the substance isregistered in the respective region, for example in order to terminatethe registering in favor of a new attempt if it is not found that theintensity comes from only a single molecule. This is very important inso far as the readout of a sensor array is often the rate-limitingfactor for concluding a cycle of the new method. The sensor array mustbe read out before the switching signal can be used to make a newselection of molecules of the substance, which are then in the statedelivering the measurement signal, so that the measurement signal cancome from this new selection of molecules. Sensor arrays suitable forcarrying out the new method and the new fluorescent light microscopecomprise CCD and preferably CMOS sensor arrays of conventional design,although when choosing these, besides the possibility of fast readout,it is necessary to ensure that the dark noise and readout noise aresmall enough to obtain a good signal-to-noise ratio when carrying outthe new method.

A fluorescence microscope for carrying out the new method of the firstaspect may furthermore comprise a separate switchback signal source forapplying a switchback signal to the specimen. It has however already beindicated in the description of the new method that the substance, withwhich these specimen's structure of interest is marked, may also returnfrom its second state to its first state spontaneously, i.e. by thermalexcitation, or that this transition may also be triggered by theexcitation signal with which the substance is primarily excited to emitthe measurement signal.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be understood better with reference to the followingdrawings. The parts in the drawings are not necessarily represented trueto scale; rather, emphasis is placed on it clearly illustrating theprinciples of the present invention. In the drawings, references whichare the same denote the same parts in the various views.

FIG. 1 schematically represents the structure of a fluorescent lightmicroscope for the high spatial resolution imaging of a structure ofinterest in a specimen.

FIG. 2 schematically represents a dense uniform distribution offluorescent molecules of a substance marking the specimen's structure ofinterest and the resulting intensity distribution of the fluorescentlight over the corresponding region of a sensor array, onto which thespecimen is imaged with the fluorescent light microscope according toFIG. 1.

FIG. 3 schematically represents the intensity distribution of themeasurement signal over the sensor array for the case of a singlemolecule and two closely neighboring molecules in a region of thespecimen.

FIG. 4 is a section through the intensity distribution over the sensorarray according to FIG. 3, which corresponds to one molecule.

FIG. 5 is a section through the intensity distribution over the sensorarray according to FIG. 3, which corresponds to the two closelyneighboring molecules.

FIG. 6 schematically represents the structure of a light microscope in asecond embodiment, supplemented relative to FIG. 1 by a switchbacksignal source.

FIG. 7 schematically represents another embodiment of the fluorescentlight microscope, here supplemented with a photodetector relative toFIG. 1.

FIG. 8 shows a single molecule in the observation region of thephotodetector according to FIG. 7.

FIG. 9 schematically represents the chronological sequence of photons ofthe measurement signal, which the photodetector according to FIG. 7receives from the molecule according to FIG. 8.

FIG. 10 schematically represents two molecules in the observation regionof the photodetector according to FIG. 7.

FIG. 11 schematically represents the sequence of photons of themeasurement signal from the two molecules according to FIG. 10.

FIG. 12 schematically represents the chronological sequence of thevarious optical signals in the embodiment of the fluorescent lightmicroscope according to FIG. 1.

FIG. 13 schematically represents the chronological sequence of thevarious optical signals in the embodiment of the fluorescent lightmicroscope according to FIG. 6.

FIG. 14 schematically represents the structure and the function of aFRET pair comprising a donor and an acceptor, which may be used as asubstance for marking the specimen's structure of interest in the newmethod; and

FIG. 15 schematically represents the marking of a single proteinmolecule with a plurality of FRET pairs according to FIG. 14.

FIG. 16 shows the structure of a further fluorescent light microscope.

FIG. 17 (A) shows an overall image, recorded by the new method, ofmicrotubuli of a PtK2 cell as the structure of interest. The structureis dyed with the dye rhodamine 6G. The medium, in which the cell islocated, is an aqueous buffer solution with glucose oxidase and catalase(50 mM Tris, pH 7.5, 10 mM NaCl, glucose oxidase (Sigma, G2133), 40μg/ml catalase (Roche Applied Science, 106810), 10% (w/v) glucose). Thenumber of individual images recorded for the overall image is 61440 withexposure times of 5 ms. The light intensity was constant at 50 kW/cm².

FIG. 17 (B) is a reconstruction which corresponds to aresolution-limited image of the same object as in FIG. 17 (A). Thereconstruction was generated by the sum of the 61440 individual images.The small image at the bottom right in (B) shows a profile at the markedpositions of FIGS. 17 (A) and (B), with the aid of which the resolutionincrease of the novel method may be seen clearly.

DETAILED DESCRIPTION

Now referring in more detail to the drawings, the fluorescent lightmicroscope 1 schematically represented in FIG. 1 is used for the highspatial resolution imaging of a structure of interest inside a specimen2. The structure of interest inside the specimen 2, which is notrepresented explicitly here, is marked with a substance whose moleculeshave two states, specifically a first in which they are not fluorescentand a second, in which they are excited by an optical excitation signal3 from an excitation signal source 4 to spontaneously emit fluorescentlight which is registered as a measurement signal 5 by a sensor array 6.The molecules of the substance can be switched between the first andsecond states by an optical-switching signal 7 from a switching signalsource 8. A control (not shown separately here) of the switching source8 is configured in such a way that it adjusts the intensity of theswitching signal 7 so that the number of molecules of the substance,with which the structure of interest in the specimen 2 is marked, whichare in the second state is only such that the spacing of the fluorescentmolecules in the second state is greater than a spatial resolution limitin the imaging of the specimen 2 onto the sensor array 6 by imagingoptics 9 (only indicated in FIG. 1). This makes it possible to determinethe locations of the molecules of the substance in the specimen 2 whichare in the second state, based on their associated intensitydistributions of the measurement signal 5 over the sensor array 6, withan accuracy which far exceeds the spatial resolution limit of theimaging and depends, besides the size of the molecules of the substance,essentially on the density of the pixels of the sensor array 6, theimaging scale and the signal-to-noise ratio. The position determinationmay even be finer than the pixel spacing of the sensor array 6. Thesemitransparent mirror 10 (again only indicated in FIG. 1) is used tosuperimpose the beam paths of the excitation signal 3 and of theswitching signal 7, or to isolate the measurement signal 5 which comesfrom the specimen. Narrowband color filters are generally also used hereso that only the measurement signal of interest 5 and no fractions ofthe excitation signal 3 or the switching signal 7, reflected by thespecimen, can be registered by the sensor array 6. A double arrow 11 infront of the specimen 2 indicates that the specimen 2 both is exposed tooptical signals 3, 7 and emits the optical measurement signal 5.

FIG. 2 schematically represents a uniform statistical distribution ofmolecules 12 of the marking substance in the specimen 2. All of themolecules 12 represented are in their fluorescent second state. Thespacing of the molecules 12 is less than the spatial resolution limit inthe imaging of the specimen 2 by the imaging optics 9 onto the sensorarray 6. Specifically, the spacing of the molecules 12 here isactually-much less than the resolution limit. This results in anintensity distribution of the measurement signal 5 over the sensor array6 which is constant apart from statistical fluctuations and noise, as issymbolized in FIG. 2 by uniform shading of the represented region of thesensor array 6. It is thus not possible to determine the location ofindividual molecules 12 from the measurement signal 5 registered by thesensor array 6. Structuring (not present in FIG. 2) of the distributionof the molecules 12 inside the specimen 2 also could only be resolved upto the spatial resolution limit of the imaging of the specimen 2 by theimaging optics 9 onto the sensor array 6 with such closely spacedmolecules 12 in the fluorescent state. In the new method, in which-onlya small fraction of the molecules 12 is ever converted into theirfluoresceable state, the density of the molecules actually present isirrelevant.

FIG. 3 schematically represents the imaging onto the sensor array 6 of aregion of the specimen 2, in which there are in total three molecules 12in the fluorescent second state. Two of the molecules 12 lie pairwiseclose together, while the distance 13 of the second molecule 12 fromthis pair is greater, and specifically greater than the spatialresolution limit in the imaging of the specimen 2 of the imaging optics9 onto the sensor array 6. The spacing of the two molecules 12 of thepair, on the other hand, is smaller than the resolution limit. Thesensor array 6 registers the fluorescent light coming from the molecules12 as two discrete intensity distributions 14 and 15. The intensitydistribution 14 corresponds to the single molecule 12, while theintensity distribution 15 corresponds to the pair of molecules 12.

Sections through the intensity distributions 14 and 15 are depicted inFIGS. 4 and 5. The intensity distributions essentially do not differ intheir shape. Both intensity distributions are in principle Airy disks.The intensity distribution 15, however, has twice as great an integralas the intensity distribution 14 and a greater width at half maximum.The location of the molecule 12 in the specimen 2 can be determined veryaccurately from the intensity distribution 14, specifically inparticular with a resolution higher than the spatial resolution limit ofthe imaging of the specimen 2 by the imaging optics 9 onto the sensorarray 6. The position in the x-y plane of the specimen 2 can be deducedfrom the lateral placement of the intensity distribution on the sensorarray 6, while the shape of the intensity distribution 14 allowsinferences about the location in the Z direction inside the specimen 2.The situation is different with the intensity distribution 15. Aposition in the specimen can admittedly also be assigned to it. This,however, is only the middle position of the two molecules 12 of thepair. The intensity distribution 15 does not reveal where the twomolecules 12 are located relative to the middle position. For thisreason, in the new method for the high spatial resolution imaging of astructure marked with the substance in the specimen 2, the fraction ofthe molecules 12 which is respectively switched by the switching signal7 according to FIG. 1 into the fluorescent second state is only so smallthat as many of the molecules 12 as possible are isolated, i.e. lie at.a spacing from neighboring molecules 12 so great that when imaged ontothe sensor array 6 they result in a discrete intensity distribution 14from which the location of the molecule 12 can respectively bedetermined exactly.

FIG. 6 schematically represents an embodiment of the fluorescent lightmicroscope 1 which is supplemented relative to the embodiment accordingto FIG. 1 by a switchback signal source 16 for applying a switchbacksignal 17 to the specimen 2. A further semitransparent mirror 10 isprovided in order to superimpose the beam path of, the opticalswitchback signal 17. The molecules of the substance in the specimen 2are deliberately brought from their second state back into their firststate by the switchback signal 17, in order to make a new selection ofmolecules with the switching signal 7 for the next round of determiningthe location of individual molecules in the specimen 2. In order todetermine the location of enough molecules to achieve representativeimaging of the structure marked with them in the specimen 2, the newmethod requires frequent repetition of the selection of individualmolecules with the switching signal 7, with the underlying transitionprobabilities ensuring ever-changing selections even if individualmolecules are selected several times. The switchback signal 17 is notcategorically required when the molecules return into their first statewithin an acceptable time by themselves i.e. by thermal excitation, orby the effect of the excitation signal 3. Otherwise, the switchbacksignal 17 is absolutely necessary.

The fluorescent light microscope 1 schematically represented in FIG. 7does not comprise the supplementary switchback signal source 16according to FIG. 6; it could however also be provided in thisembodiment. FIG. 7, however, serves to explain the additionalarrangement of a photodetector 18. The photodetector 18 is intended toregister the chronological sequence of individual photons of themeasurement signal 5, which come from a region of the specimen thatcorresponds to a plurality of pixels of the sensor array 6. Thismonitoring of the sequence of the photons is intended to be used toestablish very rapidly whether only a single molecule is in thefluorescent state in the respective region, or whether a plurality ofmolecules are emitting fluorescent light from this region. If aplurality of molecules are involved, with a small size of the regionthis is an indication that the intensity distributions of themeasurement light coming from it cannot be separated on the sensor array6, i.e. cannot be used for determining the location of individualmolecules. Accordingly, the registering of the measurement signal 5 bythe sensor array 6 may be terminated in favor of a new selection ofmolecules. with the switching signal 7. At least, readout of thecorresponding regions of the sensor array 6 can be obviated. In thiscase, for example, it is expedient to provide photosensors 18 forvarious regions of the specimen 2 or the sensor array 6 in the form of afurther array, but with a smaller number of pixels. Specifically, thephotodetector 18 in FIG. 7 is designed as a coincidence detectorarrangement, with two detector units 19 being connected in parallel withthe aid of a semitransparent mirror 10 as a beam splitter. Thecoincidence detector arrangement detects those cases in which photons ofthe measurement light 15 strike both detector units 19 chronologicallyvery close together, i.e. coincidences of photons. Such coincidencescannot occur when there is a single fluorescent molecule in the regionof the specimen 2 recorded by the photodetector 18, since a singlefluorescent molecule can only ever emit a single photon owing to itsexcitation and emission of the next photon is only possible as a resultof its next excitation, there being a minimum time between theindividual excitations.

These relationships will be explained again with the aid of FIGS. 8 to11. FIG. 8 shows a single molecule which according to FIG. 9 emitsphotons 20 of the measurement signal 5 that have a minimum time spacing21. If however the photodetector 18 according to FIG. 7 registers achronological sequence of photons 20 of the measurement signal 5, asschematically represented in FIG. 11 and in which a time spacing 22 thatis very much smaller than the spacing 21 according to FIG. 9 occursbetween two photons 20, this indicates at least two fluorescentmolecules 12 in the region from which the measurement signal 5 iscoming. This case is schematically represented in FIG. 10.

FIG. 12 is a plot of the chronological sequence of the switching signal7, the excitation signal 3 and the measurement signal 5 for theembodiment of the fluorescent light microscope according to FIG. 1. Itis to be emphasized that the signal shapes are depicted only veryschematically here and not necessarily corresponding to reality. Theintention is essentially to show the chronological sequence with whichparticular molecules of the substance are initially converted into thesecond state by the switching signal 7, and are then excited intofluorescence by the excitation signal 3. This fluorescence leads to themeasurement signal 5, which decays after the excitation signal 3 isextinguished. FIG. 12 schematically represents only a single cycle ofthe method carried out with the fluorescent light microscope 1. The nextcycle begins with the same signal sequence, as soon as the molecules ofthe substance have returned from the second state into their first stateby thermal excitation or owing to the excitation signal 3.

In the signal sequence according to FIG. 13, which corresponds to theembodiment of the fluorescent light microscope 1 according to FIG. 6,the next cycle of the method with the next switching signal 7 can followimmediately, because the switchback signal 17 which returns themolecules of the substance in each case into their first state occurs atthe end of every cycle.

FIG. 14 schematically represents a FRET pair comprising a donor 23 andan acceptor 24, which form subunits of a molecule 12. The donor 23 andthe acceptor 24 could be proteins, which are fused to form the molecule12. Specifically, the acceptor may be a protein known as Dronpa whilethe donor may be a protein of the ECFP type. The function of the FRETpair according to FIG. 14 in the new method is as follows. The acceptor24 is a photochromic and changes its absorption spectrum owing to theswitching signal 7. This shift of the absorption spectrum of theacceptor 24 leads to a change in the fluorescent behavior of the donor23. Specifically, the donor 23 fluoresces since energy transfer from thedonor to the acceptor due to excitation of the donor by the excitationsignal 3 is no longer possible because of the change in the absorptionspectrum of the acceptor 24, and deexcitation of the donor can now takeplace to an increased extent via the emission of fluorescent light i.e.the measurement signal 5.

FIG. 15 schematically represents the fact that a larger protein 25 mayalso be marked with a plurality of molecules 12 at a plurality ofpoints, for example in order to observe conformational changes of theprotein 25 such as foldings by the new method. The points at which themolecules 12, which are schematically represented in the form of a FRETpair according to FIG. 14, lie are typically closer together than apossible spatial resolution limit in the optical imaging of the protein25. In the new method, however, only one of the molecules 12 on theprotein 25 is ever converted into the fluoresceable state and then itslocation is exactly determined based on the measurement signal comingfrom it. This process is repeated many times with the selection of therespective molecule 12 whose location is determined exactly followingstatistical laws, so that with the limited number of molecules 12 on theprotein 25 all the proteins 12 are interrogated after a few repetitions,even though the selection of each individual time is only determined bytransition probabilities.

When carrying out the method according to the invention for the highspatial resolution imaging of a structure of interest in a specimen 102with the fluorescent light microscope 101 schematically represented inFIG. 16, light 103 on one wavelength (black line) from a light source104 is provided via a mirror 132 and focused by means of the lens 135into an objective 136. The light 103 is used for large-area illuminationof the entire region of interest in the specimen 102. Fluorescent light105 (gray line) from fluorescent dye in the specimen 102 is likewisecollected by an objective, in this case the same objective 6, andseparated from the light 103 by means of a dichroic mirror 110, and ifnecessary refined further by a suitable fluorescent light filter 139. Inconjunction with the objective 135, a lens 109 ensures suitable imagingof the fluorescent molecules of the fluorescent dye onto a sensor array106.

When carrying out a preferred embodiment of the method according to theinvention using the fluorescent light microscope 101, the followingsteps are performed:

First, a structure of interest in a specimen is dyed with anon-switchable fluorescent dye.

The specimen is then embedded in a suitable environment. This may forexample be PVA, or alternatively an aqueous medium (for example forliving cells) from which oxygen is extracted. This measure is generallynecessary since with modern technology and conventional fluorescentdyes, the lifetime of the dark triplets state in aqueous solutionswithout oxygen concentration reduction is not long enough to be able toseparate individual molecule events. The oxygen reduction may forexample be carried out by adding glucose oxidase and catalase.

Such aqueous buffers are widely known media for microscopy. One medium,which is also suitable in principle for living cell applications, is:

88% (v/v) Gibco-DMEM (Invitrogen Corporation, Carlsbad, California) with10 mM HEPES, 10% (v/v) glucose oxidase (5 mg/ml, Sigma, G2133), 2% (v/v)catalase (2 mg/ml, Roche Applied Science, 106810).

Sometimes, when the marking density i.e. the spatial density of thefluorescent dye is too high, a sufficient fraction of the molecules ofthe fluorescent dye must be irreversibly bleached by suitable exposureof the specimen to the light before the start of the actual measurement.In any event, a sufficiently large fraction of the molecules must bepumped from their fluorescent first state into their dark second stateby shining in the light before the start of the measurement, so that theimages of the few molecules remaining in the fluorescent state on thesensor array lie further away from one another than the resolution limiton a sensor array. Typical intensities are between 1 and 100 kW/cm²,depending on the environment and fluorescent dye. The intensitydistribution of the fluorescent light, which can be recorded by thesensor array at the start of is shining the light, shows theresolution-limited image of the structure of interest. This maysubsequently be used as a reference for a termination criterion. Inpractice, for recording the resolution-limited image of the structure ofinterest, the exposure time must sometimes be adapted to a cameracomprising the sensor array or the magnification thereof, or anintensity filter must be used since the camera will be optimized for thedetection of individual molecule signals. As an alternative, a lightsignal of lower intensity may also be shone in order to record adiffraction-limited reference image, before the light signal which isused to convert the multiplicity of the molecules into the dark state.

The actual measurement can be started without delay once a sufficientfraction of the molecules has been pumped into the dark state, and inany event this must be done within a period of time which is muchshorter than the lifetime of the dark state. The exposure time of theindividual images is dictated by the average time over which a molecule,which is in the luminous first state, emits fluorescent light before itis converted back into the dark second state. In the examples used, thisleads to a typical exposure time of from 2 to 10 ms. During this time,on average an order of magnitude of 1000 photons per molecule arerecorded on the detector, before it is converted back into the darkstate.

During the measurement, after molecules have been lost by irreversiblebleaching, the intensity of the light may be reduced in order to achievean optimal density of the molecules which are in the first state.

The duration of the entire measurement is dictated by the number ofindividual images and their exposure time. The number of individualimages required is dictated by the selected termination criterion. Formore complex structures, typically up to 100,000 images individual arerecorded. The total recording time is therefore of the order of minutes.

1. A method for the high spatial resolution imaging of a structure ofinterest in a specimen, having the steps: selecting a substance from agroup of substances which can be converted repeatedly by a switchingsignal from a first state having first optical properties into a secondstate having second optical properties, and which can return from thesecond state into the first state; marking the specimen's structure ofinterest with molecules of the substance; applying an intensity of theswitching signal to the specimen in order to convert fractions of thesubstance into the second state by the switching signal, the intensityof the switching signal being set so that at least 10% of the moleculesof the substance respectively remaining in the first state lie at adistance from their closest neighboring molecules in the first statewhich is greater than the spatial resolution limit of the imaging of thespecimen onto the sensor array; imaging the specimen onto a sensorarray, a spatial resolution limit of the imaging being greater (i.e.worse) than an average spacing between closest neighboring molecules ofthe substance in the specimen; using the sensor array to register anoptical signal which comes from the fraction of the substancerespectively remaining in the first state, in order to record adistribution of the measurement signal over the sensor array; anddetermining the position in the specimen of the molecules ofthe-substance respectively remaining in the-first state, which lie at adistance from their closest neighboring molecules in the first statewhich is greater than the spatial resolution limit of the imaging of thespecimen onto the sensor array, from the distribution of the measurementsignal over the sensor array.
 2. The method as claimed in claim 1,wherein the intensity of the switching signal when converting thealternating fractions of the substance into the second state is adjustedso that at least 33% of the molecules of the substance respectivelyremaining in the first state lie at a distance from their closestneighboring molecules in the first state which is greater than thespatial resolution limit of the imaging of the specimen onto the sensorarray.
 3. The method as claimed in claim 2, wherein the intensity of theswitching signal when converting the alternating fractions of thesubstance into the second state is adjusted so that at least 66% of themolecules of the substance respectively remaining in the first state lieat a distance from their closest neighboring molecules in the firststate which is greater than the spatial resolution limit of the imagingof the specimen onto the sensor array.
 4. The method as claimed in claim1, wherein the intensity of the switching signal when converting thealternating fractions of the substance into the second state is adjustedso that at least 90% of the molecules of the substance respectivelyremaining in the first state lie at a distance from their closestneighboring molecules in the first state which is greater than thespatial resolution limit of the imaging of the specimen onto the sensorarray.
 5. The method as claimed in claim 1, wherein the intensity of theswitching signal when converting the alternating fractions of thesubstance into the second state is adjusted so that essentially all ofthe molecules of the substance respectively remaining in the first statelie at a distance from their closest neighboring molecules in the firststate which is greater than the spatial resolution limit of the imagingof the specimen onto the sensor array.
 6. The method as claimed in claim1, wherein the intensity of the switching signal is set to a constantvalue over a region which has dimensions larger than the spatialresolution limit of the imaging of the specimen onto the sensor array.7. The method as claimed in claim 6, wherein the constant value isestablished as a function of a local concentration of the substance inthe specimen.
 8. The method as claimed in claim 7, wherein the localconcentration of the substance in the specimen is determined when alarger fraction of the molecules of the substance are still in the firststate.
 9. The method as claimed in claim 7, wherein the localconcentration of the substance in the specimen is determined beforehandwhen essentially all molecules of the substance in the first state. 10.The method as claimed in claim 1, wherein a region of the specimen whichcorresponds to a plurality of pixels of the sensor array is imaged ontoa photodetector, in order to observe the chronological sequence of theemission of individual photons from the region.
 11. The method asclaimed in claim 1, wherein the substance is selected from a subgroup ofsubstances which can be excited in the first state by an opticalexcitation signal to spontaneously emit fluorescent light, which isregistered as an optical measurement signal by the sensor array.
 12. Themethod as claimed in claim 11, characterized in that the substance isswitchable.
 13. The method as claimed in claim 12, wherein the substanceis selected from a subgroup of substances which can be converted fromthe second state back into the first state by an optical switchbacksignal.
 14. The method as claimed in claim 1, wherein when marking thespecimen's structure of interest with molecules of the substance, themolecules of the substance are expressed by gene technology togetherwith protein molecules of the structure of interest in the specimen. 15.The method as claimed in claim 1, wherein when marking the specimen'sstructure of interest with molecules of. the substance, binding sitesfor molecules of the substance are expressed by gene technology togetherwith protein molecules of the structure of interest in the specimen. 16.The method as claimed in claim 1, wherein a protein molecule in thespecimen is marked at a plurality of different points with molecules ofthe substance.
 17. The method as claimed in claim 1, which is carriedout in parallel with molecules of at least two different substances,from which distinguishable optical measurement signals come in theirrespective first state.
 18. A method for the high spatial resolutionimaging of a structure of interest in a specimen, having the steps:selecting a substance from a group of substances, which have a firststate with first fluorescent properties and a second state with secondfluorescent properties; which can be excited by light of one wavelengthto spontaneously emit fluorescent light; which can be converted from thefirst state into their second state by the light of the one wavelengthand which can return from their second state into their first state;marking the specimen's structure of interest with molecules of thesubstance; imaging the specimen onto a sensor array, a spatialresolution limit of the imaging being greater (i.e. worse) than anaverage spacing between closest neighboring molecules of the substancein the specimen; exposing the specimen to the light of the onewavelength in a region which has dimensions larger than the spatialresolution limit of the imaging of the specimen onto the sensor array,fractions of the molecules of the substance alternately being excited bythe light of the one wavelength to spontaneously emit fluorescent lightand being converted into their second state, and at least 10% of themolecules of the substance that are respectively in the first statelying at a distance from their closest neighboring molecules in thefirst state which is greater than the spatial resolution limit of theimaging of the specimen onto the sensor array; registering thefluorescent light which is spontaneously emitted from the region ofmolecules of the substance, in a plurality of images recorded by thesensor array during continued exposure of the specimen to the light ofthe one wavelength; and determining the position in the specimen of themolecules of the substance that are respectively in the first state,which lie at a distance from their closest neighboring molecules in thefirst state which is greater than the spatial resolution limit of theimaging of the specimen onto the sensor array, from the images recordedby the sensor array.
 19. The method as claimed in claim 18, wherein atthe start of the exposure of the specimen to the light of the onewavelength, its intensity is set to be so large that the substance isconverted into its second state until more than 90% of the molecules ofthe substance have been converted into the second electronic state. 20.The method as claimed in claim 18, wherein at the start of the exposureof the specimen to the light of the one wavelength, its intensity is setto be so large that the substance is converted into its second stateuntil essentially all of the molecules of the substance have beenconverted into the second electronic state.
 21. The method as claimed inclaim 17, wherein the substance is selected from a subgroup ofsubstances which comprises such substances as return spontaneously fromtheir second state into their first state; return from their secondstate into their first state by the action of the light of the onewavelength; and return into their first state spontaneously and byaction of the light of the one wavelength.
 22. The method as claimed inclaim 21, wherein the first state and the second state are differentelectronic states of the substance.
 23. The method as claimed in claim22, wherein the substance is not switchable.
 24. The method as claimedin claim 22, wherein the first state is a singlet state and the secondstate is a triplet state of the substance.
 25. The method as claimed inclaim 22, wherein at least one measure is implemented which modifies thelifetime of the second state of the fluorescent dye in the specimen. 26.The method as claimed in claim 22, wherein at least one measure isimplemented which extends the lifetime of the second state of thefluorescent dye in the specimen.
 27. The method as claimed in claim 22,wherein before the images are recorded, a fraction of the substance isconverted by photobleaching by means of a high intensity of light, whichis selected from the light of the one wavelength and the light ofanother wavelength, into a persistent dark state which differs from thefirst state and the second state.
 28. The method as claimed in claim 17,wherein the intensity of the light of the one wavelength is set to aconstant value during the recording of the images.
 29. The method asclaimed in claim 17, wherein the intensity of the light of the onewavelength is set to an intensity profile, time-modulated with thesequence of the recording of the images, during the recording of theimages.
 30. The method as claimed in claim 17, the light of the onewavelength is directed continuously onto the region of the specimen. 31.The method as claimed in claim 17, wherein the light of the onewavelength is directed onto the region of the specimen in rapid pulseswhich are not resolved during the recording of the images.
 32. Themethod as claimed in claim 17, wherein the individual recorded imagesare evaluated online as to whether they show inseparable fluorescentmolecules of the substance, and in that the intensity of the light isvaried until a density threshold for such inseparable fluorescentmolecules is fallen below.
 33. The method as claimed in claim 17,wherein the individual recorded images are evaluated online in respectof the maximum density in which they show separable fluorescentmolecules of the substance, and in that the intensity of the light isvaried until a density threshold for such separable fluorescentmolecules is reached.
 34. The method as claimed in claim 17, at thestart of exposing the specimen to the light of the one wavelength, anintensity distribution of the fluorescent light of the entire substancein the specimen is recorded by the sensor array with the spatialresolution of the imaging of the specimen onto the sensor array.
 35. Themethod as claimed in claim 34, wherein a termination criterion for therecording of further images of the same region of the specimen isdefined on the basis of the intensity distribution of the fluorescentlight of the entire substance in the specimen.
 36. The method as claimedin claim 35, wherein each position of a molecule of the substanceregistered in the mutually successive images convoluted with the PSF(Point Spread Function) of the imaging of the specimen onto the sensorarray, and this reconstruction is compared with the initially recordedintensity distribution.
 37. The method as claimed in claim 17, whereinthe specimen's structure of interest is marked with the substance bymodifying a biological specimen with gene technology so that it. itselfexpresses the substance.
 38. The method as claimed in claim 17, whereinthe specimen's structure of interest is marked with the substance bymodifying a biological specimen with gene technology so that itexpresses proteins with specific binding sites for the substance. 39.The method as claimed in claim 17, wherein the specimen's structure ofinterest is marked with the substance by modifying a biological specimenwith gene technology so that it expresses proteins with specific bindingsites for a linker coupled to the substance.